This research will develop, characterize and optimize a novel synthetic gene delivery system for the delivery of the human cystic fibrosis transmembrane conductance regulator encoding DNA to human airway epithelial and submucosal cells in the lung. The delivery system consists of a targeting ligand, membrane destabilizing component, nuclear entry component and DNA condensor that form a complex directly with the DNA. The targeting ligand is attached to the DNA via a DNA intercalator. The current intercalator is a bis-acridine chemically linked so that the acridines have the correct geometry for bis-intercalation. In addition the intercalator has a spacer arm terminating with a sulfhydryl reactive moiety so that peptide or protein ligands for cell surface receptors can be easily attached to the DNA. The membrane destabilizing component is a cationic, amphipathic decapeptide that can bind to the DNA and in the presence of the phospholipid phosphatidylethanolamine facilitates transmembrane transfer of DNA. The peptide DNA-lipid complex is between 20 to 100 times more effective than the cationic lipid DOTMA at mediating transfection of cells in culture. The research plan has four interacting components: 1. Synthesis of the delivery ligands and membrane destabilizers 2. Optimization and characterization of the transfection system in cultured cells and rat lung using reporter genes, 3. Transfection of CFTR into cell lines and cultured human primary lung cells and assessment of function. 4. Transfection of CFTR into rat lung and characterization of the extent, duration and location of expression of human CFTR in the rat. A major emphasis of the research is on the synthesis and detailed physico- chemical characterization of the delivery complex in order to correlate the various components with successful transfection. The pulmonary surfactant protein SP-A will be attached to the complex to exploit receptor mediated endocytosis of SP-A to accelerate entry of the DNA complex into airway epithelial cells. Since transfection frequency may be limited by nuclear entry of DNA. Nuclear localization peptide-acridine conjugates will be attached to DNA to increase nuclear uptake. The influence of increased plasmid nuclear localization on gene expression will be quantitated. The level of expression of transfected human CFTR gene in non-expressing human cells will be estimated by Northern blots and antibody staining. More importantly the function of the transfected CFTR will be quantitated in human Hela and HL60 cells in culture using chloride sensitive fluorescent indicators. Function will also be measured in human CF primary cultures of epithelial and tracheobronchial glands on supported monolayers using Ussing chambers. Our goal is to understand which chemical and/or physico-chemical factors of the delivery complex are required for high level transfection and expression of CFTR in human epithelial and submucosal cells with the ultimate goal of a non-viral gene therapy treatment for Cystic Fibrosis.